Cloning and in silico analysis of a cinnamyl alcohol dehydrogenase gene in Pennisetum purpureum

Abstract

Lignin is a major constituent of plant cell walls and indispensable to the normal growth of a plant. However, the presence of lignin complicates the structure of the plant cell walls and negatively influences pulping industry, lignocellulose utilization as well as forage properties. Cinnamyl alcohol dehydrogenase (CAD), a key enzyme involved in lignin biosynthesis, catalyses the last step in monolignol synthesis and has a major role in genetic regulation of lignin production. In the present study, a 1 342-bp cDNA fragment of CAD gene, named PpCAD, was isolated from Pennisetum purpureum using strategies of homologous clone and rapid amplification of cDNA end. It was translated into an intact protein sequence including 366 amino acid residues by ORF Finder. The genomic full-length DNA of PpCAD was a 3 738-bp sequence containing four exons and three introns, among which the 114-bp exon was considered to be a conserved region compared with other CADs. Basic bioinformatic analysis presumed that the PpCAD was a nonsecretory and hydrophobic protein with five possible transmembrane helices. The phylogenetic analysis indicated that the PpCAD belonged to the class of bona fide CADs involved in lignin synthesis and it showed a high similarity (nearly 90%) with CAD protein sequences of Sorghum bicolor, Panicum virgatum and Zea mays in Gramineae. Furthere, PpCAD amino acid sequence was demonstrated to have some conserved motifs such as Zn-binding site, Zn-catalytic centre and NADP(H) binding domain after aligning with other bona fide CADs. Three-dimensional homology modelling of PpCAD showed that the protein had some exclusive features of bona fide CADs.

Characterization of Cinnamyl Alcohol Dehydrogenase gene family in lignifying tissues of Tectona grandis L.f.

“…Elements of the secondary structure, domains and relevant residues, catalytic and structural sites were found following the annotations described by Tang et al (Tang et al 2014a), Jin et al (Jin et al 2014), Larroy et al (Larroy et al 2002) and Youn et al (Youn et al 2006 (Guo et al 2010) and Barakat et al (Barakat et al 2010), and groups (colored) were defined following the methods of Barakat et al . Secondary structure elements, important relevant residues, and catalytic and structural sites were found according to the methods described by Tang et al (Tang et al 2014a), Jin et al (Jin et al 2014), Larroy et al (Larroy et al 2002) and Youn et al (Youn et al 2006) annotations. Peptide signals and protein subcellular localization was performed following the method described by Jin et al (Jin et al 2014), using Signal IP4.1 program (http://www.cbs.dtu.dk/services/ SignalP/) and CELLO program v.2.5 (http://cello.life.nctu.edu.…”

“…White star indicates key Phe299 residue for substrate specificity and black star indicates key Asp123 residue for substrate binding. Most of the alignment information was identified according to the results described by Jin et al (Jin et al 2014) and Tang et al (Tang et al 2014a). Figure 3 Phylogenetic tree of monocots, dicots and Tectona grandis CAD proteins.…”

“…Lignin, a three-dimensional phenolic heteropolymer composted by p-coumaryl (H), coniferyl (G) and sinapyl (S) alcohols has essential roles in plant structural rigidity, pathogen defense and conduction of water and nutrients (Bonawitz and Chapple 2010;Xu et al 2013;Tang et al 2014a). It is the major component of secondary cell walls, for cells that will die; it constitutes the last step of cell division, expansion and elongation before cell death (Eudes et al 2014;Laurichesse and Avérous 2014;Zeng et al 2014).…”

“…In the last decade, lignin has been a target for several studies due to its agricultural and economic importance, suggesting that at least ten enzymes are required for lignin biosynthesis . Lignin is the final product of the phenylpropanoid pathway, with cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) being the enzyme that catalyzes the conversion of cinnamyl aldehydes to cinnamyl alcohols (Li et al 2006;Tang et al 2014a).…”

“…Brachypodium (Bukh et al 2012), camellia, (Deng et al 2013) and melon (Jin et al 2014) also present several CAD genes described. Nearly, 80 CAD genes in 35 plants have been studied, usually existing in a multi-gene family and with differential expression during plant development and environmental conditions (Guo et al 2010;Tang et al 2014a).…”